Desmoglein-2 carrying cancer stem cell-derived extracellular vesicles promote lung cancer aggressiveness

Lung cancer remains the leading cause of cancer-related deaths worldwide, with non-small cell lung cancer (NSCLC) accounting for approximately 85% of all cases. Despite advances in targeted and immuno-therapies, overall survival for NSCLC remains below 25%, due to late-stage detection and therapeutic resistance. Cancer stem cells (CSCs), a highly tumorigenic subpopulation within NSCLC, drive relapse, metastasis, and treatment failure. Understanding the mechanisms by which CSCs promote the aggressiveness of NSCLC may reveal new diagnostic and therapeutic opportunities. CSCs establish an intercellular communication network through the secretion of extracellular vesicles (EVs), membrane-bound nanoparticles that transport proteins, lipids, and nucleic acids that can influence recipient cell behavior. Our previous work demonstrated that CSCs secrete significantly more EVs compared to bulk cancer cells (BCCs) and induce an enhanced tumorigenic phenotype in BCCs. However, the EV protein cargo that mediates these CSC-driven phenotypes and the signaling they promote remain poorly understood. This study aimed to identify and characterize CSC-derived EV protein cargo that contributes to NSCLC growth and metastasis. CSC- and BCC-derived EVs were isolated using tangential flow filtration and characterized according to MISEV guidelines. Mass spectrometry and pathway analysis were performed to compare the proteome of CSC- and BCC-derived EVs, followed by functional characterization in vitro and in vivo. Our results showed that EVs exhibited expected morphological, biochemical, and physical properties. Pathway analyses showed that CSC-derived EVs were enriched in proteins that localize to exosomes and associate with metabolism and cell adhesion. Comparison of BCC and CSC-derived EVs revealed Desmoglein-2 (DSG2) as one of the proteins exclusively detected in CSC-derived EVs. shRNA-mediated knockdown of DSG2 significantly reduced invasion, clonal expansion, and transformed growth in vitro and decreased tumor growth and metastasis in vivo . Furthermore, DSG2-competent EVs partially rescued the effects of DSG2 knockdown on CSC tumor growth in vivo . Together, these findings identify DSG2 as a key CSC-specific EV protein cargo that promotes an enhanced tumorigenic phenotype and NSCLC tumor growth. This work highlights the role of CSC-derived EV proteins as mediators of NSCLC aggressiveness and as candidates for biomarker and therapeutic development.